A variety of mutant strains of mouse L cells have been isolated which are either respiration-deficient or resistant to drugs which inhibit mitochondrial function such as oligomycin, antimycin A and chloramphenicol. Many of these mutations are cytoplasmically-determined and are therefore thought to be sequence alterations in mitochondrial DNA (mtDNA). In order to prove this hypothesis and to construct a genetic map of mtDNA we will attempt to establish correlations between the biochemical phenotype of each mutant and the specific mtDNA sequence which is altered. Specifically, we will determine which mtDNA gene products are missing or defective in the respiratory-deficient mutants and map the sites of lesions in their mtDNAS. We will determine the sequence of the large ribosomal RNA (16S RNA) gene in normal and chloramphenicol-resistant L cells. These sequences are contained in recombinant plasmids which we have constructed using mtDNA, from normal and chloramphenicol-resistant cells. We will also isolate the dicyclohexylcarbodiimide (DCCD) binding protein from the mitochondrial ATPase complex and determine its sequence in normal and oligomycin-resistant L-cell lines.